Cryo-electron microscopy structures of human thyroid peroxidase (TPO) in complex with TPO antibodies.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the hTPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM. The two structures (global resolution 3.92 and 3.4 Å for the 2G4 complex and 4F5 complex, respectively) show TPO as a monomer with four domains; the N-terminal domain, the peroxidase domain (POD), the complement control protein (CCP)-like domain and the epidermal growth factor-like domain which are all visible in the structures. The relative positions of the domains are fixed with a disulphide bond between cysteine residues Cys146 in the POD and Cys756 in the CCP domain preventing significant flexibility of the molecule. The entrance to the enzyme active site, the haem group and the calcium binding site are clearly visible on the opposite side of the TPO molecule from the 2G4 and 4F5 binding sites. Extensive interactions are seen between TPO and the two antibodies which both bind to distinct epitopes on the POD domain, including some residues in the immunodominant region B mainly via different residues. However, the epitopes of the two antibodies contain three shared TPO residues. This is the first high-resolution structure of TPO to be reported and it should help guide the development of new inhibitors of TPO enzyme activity for therapeutic applications.

Distinct features of cap binding by eIF4E1b proteins.

eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m(7)GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α+ß fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N(7) of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N(7) position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.

TSH receptor monoclonal antibodies with agonist, antagonist, and inverse agonist activities.

Autoantibodies in autoimmune thyroid disease (AITD) bind to the TSH receptor (TSHR) and can act as either agonists, mimicking the biological activity of TSH, or as antagonists inhibiting the action of TSH. Furthermore, some antibodies with antagonist activity can also inhibit the constitutive activity of the TSHR, that is, act as inverse agonists. The production of animal TSHR monoclonal antibodies (MAbs) with the characteristics of patient autoantibodies and the isolation of human autoantibodies from patients with AITD has allowed us to analyze the interactions of these antibodies with the TSHR at the molecular level. In the case of animal MAbs, advances such as DNA immunization allowed the production of the first MAbs which showed the characteristics of human TSHR autoantibodies (TRAbs). Mouse MAbs (TSMAbs 1-3) and a hamster MAb (MS-1) were obtained that acted as TSHR agonists with the ability to stimulate cyclic AMP production in CHO cells expressing the TSHR. In addition, a mouse TSHR MAb (MAb-B2) that had the ability to act as an antagonist of TRAbs and TSH was isolated and characterized. Also, a mouse TSHR MAb that showed TSH antagonist and TSHR inverse agonist activity (CS-17) was described. Furthermore, a panel of human TRAbs has been obtained from the peripheral blood lymphocytes of patients with AITD and extensively characterized. These MAbs have all the characteristics of TRAbs and are active at ng/mL levels. To date, two human MAbs with TSHR agonist activity (M22 and K1-18), one human MAb with TSHR antagonist activity (K1-70) and one human MAb (5C9) with both TSHR antagonist and TSHR inverse agonist activity have been isolated. Early experiments showed that the binding sites for TSH and for TRAbs with thyroid stimulating or blocking activities were located on the extracellular domain of the TSHR. Extensive studies using TSHRs with single amino acid mutations identified TSHR residues that were important for binding and biological activity of TSHR MAbs (human and animal) and TSH. The structures of several TSHR MAb Fab fragments were solved by X-ray crystallography and provided details of the topography of the antigen binding sites of antibodies with either agonist or antagonist activity. Furthermore stable complexes of the leucine-rich repeat domain (LRD) of the TSHR with a human MAb (M22) with agonist activity and with a human MAb (K1-70) with antagonist activity have been produced and their structures solved by X-ray crystallography at 2.55 and 1.9Å resolution, respectively. Together these experiments have given detailed insights into the interactions of antibodies with different biological activities (agonist, antagonist, and inverse agonist) with the TSHR. Although the nature of ligand binding to the TSHR is now understood in some detail, it is far from clear how these initial interactions lead to functional effects on activation or inactivation of the receptor.

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

Targeting LMO2 with a peptide aptamer establishes a necessary function in overt T-cell neoplasia.

LMO2 is a transcription regulator involved in human T-cell leukemia, including some occurring in X-SCID gene therapy trials, and in B-cell lymphomas and prostate cancer. LMO2 functions in transcription complexes via protein-protein interactions involving two LIM domains and causes a preleukemic T-cell development blockade followed by clonal tumors. Therefore, LMO2 is necessary but not sufficient for overt neoplasias, which must undergo additional mutations before frank malignancy. An open question is the importance of LMO2 in tumor development as opposed to sustaining cancer. We have addressed this using a peptide aptamer that binds to the second LIM domain of the LMO2 protein and disrupts its function. This specificity is mediated by a conserved Cys-Cys motif, which is similar to the zinc-binding LIM domains. The peptide inhibits Lmo2 function in a mouse T-cell tumor transplantation assay by preventing Lmo2-dependent T-cell neoplasia. Lmo2 is, therefore, required for sustained T-cell tumor growth, in addition to its preleukemic effect. Interference with LMO2 complexes is a strategy for controlling LMO2-mediated cancers, and the finger structure of LMO2 is an explicit focus for drug development.

A mutation in helicase motif IV of herpes simplex virus type 1 UL5 that results in reduced growth in vitro and lower virulence in a murine infection model is related to the predicted helicase structure.

A variant was selected from a clinical isolate of herpes simplex virus type 1 (HSV-1) during a single passage in the presence of a helicase-primase inhibitor (HPI) at eight times the IC(50). The variant was approximately 40-fold resistant to the HPI BAY 57-1293 and it showed significantly reduced growth in tissue culture with a concomitant reduction in virulence in a murine infection model. The variant contained a single mutation (Asn342Lys) in the UL5 predicted functional helicase motif IV. The Asn342Lys mutation was transferred to a laboratory strain, PDK cl-1, and the recombinant acquired the expected resistance and reduced growth characteristics. Comparative modelling and docking studies predicted the Asn342 position to be physically distant from the HPI interaction pocket formed by UL5 and UL52 (primase). We suggest that this mutation results in steric/allosteric modification of the HPI-binding pocket, conferring an indirect resistance to the HPI. Slower growth and moderately reduced virulence suggest that this mutation might also interfere with the helicase-primase activity.

Substrate specificity and molecular modelling of the feline herpesvirus-1 thymidine kinase.

Feline herpesvirus-1 (FHV-1) causes a severe upper respiratory and ocular disease in cats. An effective antiviral compound is required for treating FHV-1 infections. The virus-encoded thymidine kinase (TK) is the molecular basis for selective activation of commonly used antiviral nucleoside analogue drugs, e.g. acyclovir (ACV), penciclovir (PCV) and ganciclovir (GCV). The substrate specificity of a recombinant FHV-1 TK, expressed in Escherichia coli, was studied. FHV-1 TK efficiently phosphorylated its natural substrate deoxythymidine. However, it exhibited relatively lower affinity for the guanosine analogue substrates. PCV was most efficiently phosphorylated, followed by GCV, with approximately twofold reduction in the phosphorylation rate. The lowest phosphorylation rate was recorded for ACV. To correlate these biochemical data with structural features of the FHV-1 TK, a three-dimensional (3D) model of this enzyme was constructed based on sequence homology with two other herpesviral TKs, encoded by equine herpesvirus-4 (EHV-4) and herpes simplex-1 (HSV-1). Mutational analysis of the amino acids forming the FHV-1 TK active site identified two residues (Y29 and F144) as being critical for the differential ability of this enzyme to phosphorylate nucleoside analogues. A double substitution of Y29H/F144Y resulted in a threefold increase in the ACV phosphorylation rate.

Molecular interactions between the TSH receptor and a Thyroid-stimulating monoclonal autoantibody.

OBJECTIVE: To study the molecular interactions between the thyroid-stimulating hormone (TSH) receptor (TSHR) and a human thyroid-stimulating monoclonal autoantibody (M22). DESIGN: Amino acid mutations were introduced in the variable region gene sequences of M22 and the wild-type (WT) or mutated M22 Fab expressed in Escherichia coli. The ability of WT or mutated M22 Fab to inhibit binding of (125)I-TSH or (125)I-M22 to the TSHR and to stimulate cyclic adenosine monophosphate (AMP) production in Chinese hamster ovary cells expressing WT TSHRs was studied. Mutated TSHRs were also used in these studies in combination with WT or mutated M22 Fab to further identify interacting residues in the TSHR-M22 complex. MAIN OUTCOME: Out of 11 amino acid changes in the heavy chain (HC) of M22, 7 had an effect on M22 Fab biological activity, while in the case of 1 mutation the Fab was not expressed. In particular, stimulating activity of M22 Fab mutated at HC residues, D52, D54, and Y56 was markedly reduced. Mutation of M22 light chain (LC) D52 also reduced M22 Fab stimulating activity, while mutations at two further residues (LC D51 and LC D93) showed no effect. Reverse charge mutations at M22 HC D52 and TSHR R80 provided experimental evidence that these two residues interacted strongly with each other. CONCLUSION: Mutation of both the TSHR and M22 Fab has allowed identification of some residues critical for the receptor-autoantibody interaction. This approach should lead to detailed mapping of the amino acids important for M22 biological activity.

Crystal structure of the TSH receptor in complex with a thyroid-stimulating autoantibody.

OBJECTIVE: To analyze interactions between the thyroid-stimulating hormone receptor (TSHR) and a thyroid-stimulating human monoclonal autoantibody (M22) at the molecular level. DESIGN: A complex of part of the TSHR extracellular domain (amino acids 1-260; TSHR260) bound to M22 Fab was prepared and purified. Crystals suitable for X-ray diffraction analysis were obtained and the structure solved at 2.55 A resolution. MAIN OUTCOME: TSHR260 comprises of a curved helical tube and M22 Fab clasps its concave surface at 90 degrees to the tube length axis. The interface buried in the complex is large (2,500 A(2)) and an extensive network of ionic, polar, and hydrophobic bonding is involved in the interaction. There is virtually no movement in the atoms of M22 residues on the binding interface compared to unbound M22 consistent with "lock and key" binding. Mutation of residues showing strong interactions in the structure influenced M22 activity, indicating that the binding detail observed in the complex reflects interactions of M22 with intact, functionally active TSHR. The receptor-binding arrangements of the autoantibody are very similar to those reported for follicle-stimulating hormone (FSH) binding to the FSH receptor (amino acids 1-268) and consequently to those of TSH itself. CONCLUSIONS: It is remarkable that the thyroid-stimulating autoantibody shows almost identical receptor-binding features to TSH although the structures and origins of these two ligands are very different. Furthermore, our structure of the TSHR and its complex with M22 provide foundations for developing new strategies to understand and control both glycoprotein hormone receptor activation and the autoimmune response to the TSHR.

Recognition and cooperation between the ATP-dependent RNA helicase RhlB and ribonuclease RNase E.

The Escherichia coli protein RhlB is an ATP-dependent motor that unfolds structured RNA for destruction by partner ribonucleases. In E. coli, and probably many other related gamma-proteobacteria, RhlB associates with the essential endoribonuclease RNase E as part of the multi-enzyme RNA degradosome assembly. The interaction with RNase E boosts RhlB's ATPase activity by an order of magnitude. Here, we examine the origins and implications of this effect. The location of the interaction sites on both RNase E and RhlB are refined and analysed using limited protease digestion, domain cross-linking and homology modelling. These data indicate that RhlB's carboxy-terminal RecA-like domain engages a segment of RNase E that is no greater than 64 residues. The interaction between RhlB and RNase E has two important consequences: first, the interaction itself stimulates the unwinding and ATPase activities of RhlB; second, RhlB gains proximity to two RNA-binding sites on RNase E, with which it cooperates to unwind RNA. Our homology model identifies a pattern of residues in RhlB that may be key for recognition of RNase E and which may communicate the activating effects. Our data also suggest that the association with RNase E may partially repress the RNA-binding activity of RhlB. This repression may in fact permit the interplay of the helicase and adjacent RNA binding segments as part of a process that steers substrates to either processing or destruction, depending on context, within the RNA degradosome assembly.

The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition.

BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.

Effects of TSH receptor mutations on binding and biological activity of monoclonal antibodies and TSH.

The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling.

OBJECTIVE: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. DESIGN: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH-21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. METHODS: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. RESULTS: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% +/- 24% (n = 6), 86.7% +/- 7.6% (n = 6) and 86.5% +/- 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 +/- 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. CONCLUSIONS: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

Inhibition studies on salicylate synthase.

Analogues of chorismate and isochorismate were designed and tested as potential inhibitors in the first inhibition study against a salicylate synthase.

A model of a transmembrane drug-efflux pump from Gram-negative bacteria.

In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric inner-membrane proton-antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.

Sequence patterns derived from the automated prediction of functional residues in structurally-aligned homologous protein families.

MOTIVATION: Most proteins have evolved to perform specific functions that are dependent on the adoption of well-defined three-dimensional (3D) structures. Specific patterns of conserved residues in amino acid sequences of divergently evolved proteins are frequently observed; these may reflect evolutionary restraints arising both from the need to maintain tertiary structure and the requirement to conserve residues more directly involved in function. Databases of such sequence patterns are valuable in identifying distant homologues, in predicting function and in the study of evolution. RESULTS: A fully automated database of protein sequence patterns, Functional Protein Sequence Pattern Database (FPSPD), has been derived from the analysis of the conserved residues that are predicted to be functional in structurally aligned homologous families in the HOMSTRAD database. Environment-dependent substitution tables, evolutionary trace analysis, solvent accessibility calculations and 3D-structures were used to obtain the FPSPD. The method yielded 3584 patterns that are considered functional and 3049 patterns that are probably functional. FPSPD could be useful for assigning a protein to a homologous superfamily and thereby providing clues about function. AVAILABILITY: FPSPD is available at http://www-cryst.bioc.cam.ac.uk/~fpspd/

On the structure and function of apolipoproteins: more than a family of lipid-binding proteins.

Exchangeable apolipoproteins have been the subject of intense biomedical investigation for decades. However, only in recent years the elucidation of the three-dimensional structure reported for several members of the apolipoprotein family has provided insights into their functions at a molecular level for the first time. Moreover, the role of exchangeable apolipoproteins in several cellular events distinct from lipid metabolism has recently been described. This review summarizes these contributions, which have not only allowed the identification of the apolipoprotein domains that determine substrate binding specificity and/or affinity but also the plausible molecular mechanism(s) involved.